The major goal of this project is to elucidate the precise sequence of events which results in the formation and growth of hydroxyapatite crystals in the matrix of vertebrate mineralized tissues which include dental enamel, dentin, calcified cartilage and bone. The mineralization of these tissues is carried out by specialized cells which deposit mineral ions onto a preformed organic matrix, and is characterized by strict spatial and temporal regulation of the expression of specific gene products. The only components common to all vertebrate mineralized tissues are calcium and phosphate ions and the enzyme, alkaline phosphatase. While the beginning of this investigation will focus on alkaline phosphatase and matrix vesicles, the ultimate target of the project will be to understand how events initiated at the vesicle or cell surface result in the mineralization of matrix macromolecules, principally collagens. The experiments in this proposal will investigate hypotheses which delineate distinct functional roles for cell, vesicle and matrix constituents in matrix vesicle-dependent matrix mineralization. Hypotheses will be tested concerning the importance of specific MV and matrix components, the significance of intravesicular vs. extravesicular mineral formation and the metabolism of soluble intermediates which may include calcium phosphate ion-clusters. Experiments are proposed to investigate positive and negative modulation of ion-cluster growth and development and deposition and growth embryo crystals. At present, a gap exists in our knowledge of the initial events mineralization between events which occur in and around matrix vesicles and those occur in and around collagen fibrils. This proposal is aimed at crossing that gap by discovering the chemical species and biochemical mechanisms which may bridge it. Thus, while we believe that MV are a necessary part of the calcification machinery, we are led by our investigations, as well as those of other laboratories, to focus our attention on the interaction of matrix vesicles with the matrix. With the tools in hand to facilitate this study, we are poised to join long clashing conceptions into a unified hypothesis.